RESUMO
SUMMARY: Cancer drug development remained robust in 2023. Highlights of U.S. drug approvals this year include new immunotherapies and targeted drug development in adult and pediatric patients as well as patients with rare diseases.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Criança , Estados Unidos , Linfócitos T , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Aprovação de Drogas , Imunoterapia , United States Food and Drug AdministrationRESUMO
Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximab (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Finally, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent "linker" for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.
Assuntos
Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Cetuximab , Cristalografia por Raios X , Receptores ErbB/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos/química , Peptídeos/genética , Ligação Proteica/imunologia , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Receptor ErbB-2/imunologia , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , TrastuzumabRESUMO
Therapeutic antibodies frequently cause side effects by binding antigen in non-target tissues. Here we demonstrate a novel molecular design of antibodies that addresses this problem by reversibly "masking" antibody complementarity determining regions until they reach diseased tissues containing disease-associated proteases. Specifically, two distinct single-chain Fv (scFv) fragments derived from antibodies against the epidermal growth factor receptor (cetuximab and 425) were fused a protease susceptible linker to their epitopes, which were engineered to encourage intermolecular association. Surface plasmon resonance and flow cytometry were used to confirm that the masked complex poorly interacts with native antigen, whereas protease treatment restores antigen recognition. Minimally, the "masked" scFvs possesses an eight-fold lower association with the epitope compared with the individual scFvs unmasked by proteolytic cleavage. This molecular design may have general utility for targeted release of therapeutic antibodies at disease sites.
Assuntos
Anticorpos Monoclonais/química , Afinidade de Anticorpos , Reagentes de Ligações Cruzadas/química , Desenho de Fármacos , Receptores ErbB/imunologia , Metaloproteinase 9 da Matriz/metabolismo , Oligopeptídeos/química , Pró-Fármacos/química , Proteínas Recombinantes de Fusão/química , Anticorpos de Cadeia Única/química , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Cetuximab , Regiões Determinantes de Complementaridade/química , Reagentes de Ligações Cruzadas/metabolismo , Dimerização , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Epitopos/genética , Epitopos/imunologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Hidrólise , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/metabolismo , Mutação Puntual , Pró-Fármacos/administração & dosagem , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/imunologia , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
Monoclonal antibodies (mAbs) that inhibit activation of the epidermal growth factor receptor (EGFR) have shown therapeutic potential in select malignancies including breast cancer. Here, we describe that combined use of two such mAbs, C225 (Cetuximab) and 425 (EMD55900), reduced growth and survival of EGFR overexpressing MDA-MB-468 breast cancer cells more effectively than either antibody alone. Similarly, the C225/425 antibody combination more effectively inhibited AKT and MAPK phosphorylation in MDA-MB-468 cells. Surface plasmon resonance, size exclusion chromatography and analytical ultracentrifugation demonstrated that mAbs C225 and 425 simultaneously bind to distinct antigenic epitopes on domain III of the soluble wild-type EGFR. Furthermore, neither mAb competed with the other for binding to cells expressing either wild-type EGFR or a mutant EGFR (EGFRvIII) associated with neoplasia. Mutagenesis experiments revealed that residues S460/G461 in EGFR domain III are essential components of the 425 epitope and clearly distinguish it from the EGF/ TGFalpha binding site and the C225 interaction interface. Collectively, these results support the conclusion that therapeutic EGFR blockade in cancer patients by combined use of mAbs C225 and 425 could provide advantages over the use of the two antibodies as single agents.